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ANTIBACTERIAL EFFECTS OF OZONE ON STREPTOCOCCUS MUTANS

Fernando Astorga

A thesis submitted to the Faculty of The University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master on Science in

the Department of Operative Dentistry of the School of Dentistry

Chapel Hill 2013

Approved by:

Luiz Pimenta, DDS, MS, PhD

Roland Arnold, PhD

Andre Ritter, DDS, MS, MBA

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ABSTRACT

FERNANDO ASTORGA: Antibacterial effects of ozone on

Streptococcus mutans

(Under the direction of Luiz Pimenta)

Aims: To investigate the antibacterial effect s of ozone on Streptococcus mutans in vitro. Methods: A bacteria volume of 106 CFU/ml was inoculated on human and bovine dentin specimens and in Wilkins-Chalgren broth media (WC, Difco

Laboratories. Sparks) treated with two ozone sources ( Curozone X4®, Kavo; OL80A and

OL80W, Yanco Industries), at various concentrations (2, 20, 200, 2000 and 2100 ppm)

and exposure times (from 15 s ec to 4 min). Results: Curozone X4 had no effect on

bacteria recovery, while OL80A and OL80W showed a concentration -dependent

effect on S. mutans recover on a Trypticase™ Soy Agar with 5% Sheep Blood (Difco

Laboratories. Sparks). Concentrations of 200 and 2000 ppm showed no bacteria

recovery after treatment for 4 min, while 2 and 20 ppm showed significant

reduction of the recover ed bacteria. Conclusions: This study suggests that

Curozone X4® generator is not capable of delivering ozone to would effectively

disinfect a caries lesion.

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To my mother Amarilis who taught me the pursuit of excellence in life,

wherever you are I hope you are proud

To my wife Liliana and my children Sebastian and Clementina, your love

and support made this dream come true

I love you all!

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ACKNOWLEDGEMENTS

I want to thank the Lord and Jesus Christ my savior, for giving the

opportunity of coming here and follow his plan.

To my Thesis Committee members, Dr. Luiz Pimenta, Dr. Roland Arnold, Dr.

Andree Ritter and Dr. Kenneth Sexton, for accepting me as an advisee with my

limitations and stubbornness. I am grateful for your advice, patience, time, word of

encouragement, suggestions and guidance that made this thesis be completed.

Dr. Terry Donovan, Dr. Harald Heymann and Dr. Edward Swift, it has been

an honor to be your student.

The staff of the Department of Operative Dentistry, Shannon Tate, Dayna

McNaught, Jamie Desoto, Barbara Walton, Ginger Cole and Cynthia Lambert.

I want to thanks Mr. Eric Simmons for all your help and patience while

working with me in the lab and most of all I want to thank you for your friendship.

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Kristi and Hiroko you have been the best classmates I ever had. I will never

forget these three years of sharing accomplishments, success, laugh and tears. You

will be in my heart forever. Also to the residents that made this experience even

more enjoyable Mario, Ayesha, Roopsie, Sumi, Alex, Silvia, Upoma, Vili and Clay.

The AEGD staff, Rosanna, Christy, Pam, Christina and Daryl.

All of you helped making UNC my home!

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TABLE OF CONTENTS

Page

LIST OF TABLES………...viii

LIST OF FIGURES………. ....ix

LIST OF ABBREVIATONS…….………...x

1. INTRODUCTION……… ..…1

2. OBJECTIVES………….…….………..….…8

3. MATERIALS AND METHODS….………..…9

3.1 Bacteria species and culture. ……….……….……9

3.2 Ozone generators....……….……….………..9

3.3 Effect of ozone on human dental specimens..………..…..10

3.4Effect of ozone on bovine dental specimens………..….12

3.5Bactericidal activity as loss of recoverable CFU after exposure of S. mutans inoculated agar surfaces to defined atmospheres of ozone .……..….………...….15

3.6Influence of ozone exposure of S. mutans suspended in liquid in microtiter plate wells on bacterial viability.…………..………..….16

3.7Effect of ozone in S. mutans suspended in media in exposure times of 1, 2, 3 and 4 min……….18

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3.8Effect of ozone in S. mutans suspended in media in concentrations of 106, 105, 104

and 103 for 1 min ………..………..………..….19

3.9Effect of ozone in S. mutans suspended in media and on agar under controlled conditions ……….…..…...21

4. RESULTS………...…..22

4.1 Effect ozone in S. mutans on human dental specimens………...…….……….22

4.2 Effect ozone in S. mutans on bovine dental specimens……….………22

4.3 Effect ozone in S. mutans suspended in media under controlled conditions ………..………..…23

4.4 Effect ozone in S. mutans suspended in media for 15, 30, 45 and 60 s……….………24

4.5 Effect ozone in S. mutans suspended in media for 1, 2, 3 and 4 min..……….….…24

4.6 Effect ozone in S. mutans suspended in media in concentrations of 106, 105, 104 and 103 ..……….…….24

4.7 Effect ozone in S. mutans in media and on agar……….……..25

5. DISCUSSION………..………..….………...26

6. CONCLUS IONS.……….…………..…….………..….35

7. TABLES……...………….……….………....36

8. FIGURES…….………….……….………..…..44

APPENDICES……..………….……….……… …....54

REFERENCES………..……….……….………...….55

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LIST OF TABLES

Table

1. Indication of the ozone therapy in medicine……….………...29

2. Number of bacteria recovered after the different treatment

protocols applied with the Kavo Curozone X4® ozone generator

to human dentin specimens for 60 s (log10 CFU)……….………….……….30

3. Number of bacteria recovered after the different treatment

protocols applied with the Kavo Curozone X4® ozone generator

to bovine dentin specimens for 60 s (log10 CFU)…….…….……….………..31

4. Number of bacteria recovered and killing rates after exposing

S. mutans on agar (log10 CFU/ml) to ozone in the Modular Incubator Chamber at different concentrations for 4 min in

covered and uncovered Petri dishes..………....…..….32

5. Bacterial growth as optical density (mO.D. at λ610 nm)

attained following incubation at 37oC for 18 hr after the

different treatment protocols applied with the Kavo Curozone X4®

ozone generator on 10l of 106 CFU/ml of S. mutans………….….….…..…33

6. Bacterial growth as optical density (mO.D. λ610 nm)

ozone generator on 10l of 106 CFU/ml of S. mutans…...34

ozone generator on 10l of a bacteria titration from

106 to 103 CFU/ml of S. mutans…………..……….…….35

recovery in media and recovery (log10 CFU/ml) on agar

different treatment protocols applied with the ozone generator OL80W in the Modular Incubator Chamber

at 2000 ppm for 4 min………..36

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LIST OF FIGURES

Figure

1. Curozone X4®………...………37

2. Curozone X4schematic functioning………..………...38

3. OL80A Ozone generator. ………...………..…41

4. Mouse Exposure Chamber …...………....42

5. OL80W Ozone generator. .………….……….….43

6. Modular Incubator Chamber …..…..……….………...44

7. Human root specimen………..….……...39

8. Bovine root specimen……….……..40

9. 96 well microtiter plate………...….……45

10. Experiment 10 sample distribution……….….…46

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LIST OF ABBREVIATONS

S. mutans: Streptococcus mutans E. faecalis: Enterococcus faecalis S. sobrinus: Streptococcus sobrinus L. acidophilus: Lactobacillus acidophilus L. casei: Lactobacillus casei

A. naeslundii: Actinomyces naeslundii V. dispar: Veillonella dispar

F. nucleatum: Fusobacterium nucleatum S. oralis: Streptococcus oralis

C. albicans: Candida albicans S. aureus: Staphylococcus aureus E. coli: Escherichia coli

B. atrophaeus: Bacillus atrophaeus

mg/L: Milligrams per liter

mO.D.: Millioptical density

ppm: Parts per million

TSA: Trypticase soy agar

TSB: Trypticase soy broth

V. dispar: Veillonella dispar

l: Microliter

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1. Introduction

Since the 1970’s there has been a significant reduction in dental caries in

industrialized countries for children and adolescents, due to environmental and educational

factors such as the increased use of fluoride in public water supplies, dentifrices and dental

products; improved oral hygiene and prophylaxis; dietary counseling; and increased access to

dental care.1 Nevertheless, dental caries is still a common disease, experienced by almost 80%

of children by the age of 18 and by almost 90% of adults.1

Dental caries is a multifactorial disease, characterized at the tooth level by a dynamic

imbalance of the demineralization/remineralization processes over time2. The mineral content

of the tooth is sensitive to increases in acidity. For enamel surfaces, when the pH drops below

5.5 demineralization proceeds faster than remineralization resulting in a net loss of mineral

structure. Carious lesions are caused by bacterial fermentation of dietary carbohydrates into

acids (principally lactic acid) within the dental biofilm. If the acid production and

accumulation exceeds the buffering capacity of the oral environment, demineralization

occurs eventually leading to cavitation.2

Dental biofilm is a key requirement for carious lesions to develop. Dental biofilm

results from adherence of oral microorganisms to and growth onto the tooth surface. When

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is lowered, acids diffuse rapidly into the underlying enamel or dentin.3 The two most

important groups of bacteria that predominantly produce acids are the mutans streptococci

(Streptococcus mutans and Streptococcus sobrinus) and the lactobacilli.3 These acidogenic bacteria metabolize a variety of fermentable, dietary carbohydrates to lactic acid, the strongest

naturally occurring acid produced by acidogenic bacteria during the caries process.3

Dental caries may have a significant impact on an individual’s life. The most common

consequences of untreated lesions are discomfort and pain.4 Restorative dental treatments can

now be provided pain free, apart from the pain of the local anesthetic injection. However, for

some people restorative treatments are associated with fear and anxiety, which may become

barriers to dental attendance as well as financial limitations.4 Treatment avoidance can

subsequently lead to further progression of caries lesions which, in turn, may cause more

distress and long-term complications4. Gross decay may lead to disturbances in eating and

sleeping patterns because of pain. Psychological distress can arise from the embarrassment

and self-consciousness of having missing or decayed teeth, especially in the anterior

dentition.4

According to the anatomical location of carious lesions, it is possible to differentiate

between coronal lesions, which may affect the pits and fissures or the smooth surfaces of a

tooth in enamel, and root lesions, which affect the exposed root surface in dentin.

The prevalence of root caries begins at about 30–40 years of age and tends to increase

thereafter. Adults are keeping their teeth longer however, increased retention of teeth means

that more adults are at risk for root caries. Gingival recession caused by aging puts root

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surfaces especially at risk. Among dentate adults, the prevalence of root caries experience is

about 8 times higher among those aged 75+ years (55.9%) compared to those aged 18 to 24

years (6.9%).5

Ozone has been proposed as a therapeutic agent for dental caries.6-18 Ozone (O3) is a

triatomic molecule, consisting of three oxygen atoms with a molecular weight of 47.98 g/mol.

Ozone is a highly thermodynamically unstable compound that, dependent on system

conditions such as temperature and pressure, decomposes to pure oxygen with a short

half-life.19 Ground-level ozone is an air pollutant with harmful effects on the respiratory system.

Ozone in the upper atmosphere filters potentially damaging ultraviolet light from reaching the

Earth’s surface.19

The word ozone was first introduced by Schonbein in 1840. He subjected

oxygen to electrical discharges and noted “the odor of electrical matter”. Schonbein

concluded that odor was due to a gas which he named ozone, from the Greek Ozein, which

means smell.6

Ozone, in the gaseous or aqueous phase, has been shown to be a powerful

antimicrobial agent against bacteria, fungi, protozoa, and viruses.20 It is generally accepted

that the strong oxidizing potential of ozone initiates the destruction of cell walls and

cytoplasmic membranes of bacteria and fungi. 12, 21 During this process, ozone reacts with

glycoproteins, glycolipids, and select amino acids resulting in inhibition of the enzymatic

control systems of the cell. This inhibition results in a collapse in the permeability barrier

function of the membrane, permitting the ready diffusion of ozone and other toxic molecules

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into the cell resulting in microbial death. 21 Ozone can attack many biomolecules, especially

the cysteine, methionine, and histidine amino acid residues of proteins. By oxidizing critical

biomolecules associated with the etiology of dental caries, ozone can have disruptive effects

on the cariogenic processes of bacteria.21 For example, ozone is capable of decarboxylation of

pyruvic acid to the weaker acetic acid. It has been shown that remineralization of incipient

carious lesions can be encouraged when the production of acetic acid, or other high pKa acids

found in resting biofilm, buffers plaque fluid.21

The water industry has used ozone to kill microorganisms for many years.22 One of the

most common uses of ozone is treating drinking water for disinfection; oxidation of natural

organic matter; degradation of hazardous micropollutants; algae inactivation; improvement of

color, taste, and odor of water.23

Ozone has also been tested in medicine to decontaminate hospital side rooms, rooms

contaminated with methicillin-resistant S. aureus and in auto-hemotherapy.23 Ozone has been

used in the medical field for over 150 years to disinfect and treat infections, wounds and

multiple diseases (Table 1).24 Some of the contraindications for the ozone therapy are acute

alcohol intoxication, recent myocardial infarction, hemorrhage from any organ, pregnancy,

hyperthyroidism, thrombocytopenia and ozone allergy.19

The first use of ozone in dentistry is attributed to Dr. E. Fisch who used ozonated

water in his practice;21 and Dr. Erwin Payr used it in surgery and reported his results at the

59th Congress of the German Surgical Society in Berlin (1935).21 In dental surgery, ozonated

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water was used to promote hemostasis, enhance local oxygen supply, and inhibit bacterial

proliferation.21 Theoretically, ozone in either gas or ozonated water forms, can reduce the

bacterial count in biofilm as well as in active carious lesions, and therefore it may temporarily

arrest the progression of caries, resulting in prevention of or delaying the need for tooth

restorations.21 In the dental field, ozone has been advocated for treatment in endodontics,

ozonated oils as medicaments, root and cavitated caries and for its potential application in

reducing bacterial counts in dental unit water delivery systems.15 Cesar et al. (2012) tested

the effectiveness of ozonated water for disinfection of the surfaces of dental diamond burs

that were contaminated with Eschericia coli, Staphylococcus aureus, Candida albicans, or the

spores of Bacillus atrophaeus. They found that ozonated water effectively reduced the

number of CFU/mL in an exposure of 10 or 30 min at a concentration of 10 mg/L for all

microorganisms.25

There is evidence that ozone can effectively kill microorganisms on surfaces26, 27 but

the evidence that ozone effectively kills microorganism in biofilm is contradictory. There are

reports that ozone is effective in short-time exposures (10 seconds),11, 13 while other studies

have shown its effectiveness in short20 and longer exposures (over 60 seconds).28 There are

also other authors who claim that ozone applied as gas has no effect on biofilm.22, 29, 30 Many

authors claim ozone has an anticariogenic effect not only in vitro11, 13, 14, 20 but also in vivo, and clinically by reducing the number of cariogenic bacteria in the oral cavity reversing the

caries process in existing lesions.7, 10-12 Saini (2011) advocates the use of ozone as an

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anticariogenic agent.31 It has been suggested that the application of ozone, as an adjunct

therapy in association with a remineralizing agent, could arrest or reverse carious lesions,

providing an alternative to restorative treatment.7, 8, 10, 11 Using ozone to reduce the bacterial

count in active carious lesions might temporarily modify the biofilm allowing the arrestment

of caries lesions11. This could prevent or delay the need for restorative procedures. The

clinical protocol for application of ozone is purportedly fast and easy. It has been proposed

that the application of 10 to 60 seconds of ozone could reduce the number of most of the

microorganisms in leathery (active) root caries lesions.4 These studies report that ozone acts

rapidly and at lower concentrations than those required for chlorine, and has no side-effects

such as taste and odor which are characteristic of other disinfectant agents.4 Once the

ecological niche of the acid-producing microorganisms has been reduced or eradicated, the

area is treated with a remineralizing solution. It has been suggested that for clinical treatment

the ozone exposure be repeated at 3 and 6 months after the initial application.12

Knight et al. in 2008 found that the infusion of ozone into non-carious dentin

prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four-week

period. It has been hypothesized that ozone treatment might alter the surface wettability of

dentin through reaction with organic constituents leading to a delay in bacterial colonization.14

In contrast several publications report negative results both in vitro22, 29, 30, 32 and in

clinical trials.33 Two systematic4, 21 and one Cochrane review34 agree in “given the high risk of

bias in the available studies and lack of consistency between different outcome measures,

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there is no reliable evidence that application of ozone gas to the surface of carious teeth stops

or reverses the caries process. There is a fundamental need for more evidence of appropriate

rigor and quality before the use of ozone can be accepted into mainstream primary dental care

or can be considered a viable alternative to current methods for the management and

treatment of dental caries”.34

The publication by Brazzelli et al. concluded that the current

evidence base for ozone is insufficient to say that ozone is a cost-effective addition to the

management and treatment of occlusal and root caries4. Before expensive and

time-consuming independent randomized clinical trials are performed, well-controlled in vitro

proof of concept study showing that ozone kills cariogenic bacteria are needed.

Hems et al. in 2005 showed the inability of gas ozone to effectively kill E. faecalis

after a 240 s exposure; stating that “gaseous ozone had no significant antibacterial effect on

the biofilms. This was predictable considering that the effectiveness of ozone is highest in

solution and given the previous results with biofilms, gaseous ozone would not be expected to

exert any great antibacterial effect”. They state that “Ozone is a selective oxidant and affects

only certain compounds but when it dissolves in water, it becomes highly unstable and rapidly

decomposes through a complex series of chain reactions”.22 Consequently, there is a

fundamental need for more evidence that ozone can be accepted into mainstream primary

dental care and be considered a viable alternative as adjunctive treatment of dental caries.

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2. Objectives

1. To develop a mechanical model that simulates a “lesion” in dentin as a surface for

reproducible recovery of inoculated bacteria to test efficacy of ozone delivery to target

bacteria in a relevant environment.

2. To treat that lesion model with ozone to simulate clinical conditions.

3. To determine the conditions and circumstances where ozone can have a killing effect

on S. mutans (concentration, exposure time, bacteria on surface or in media)

4. To investigate if ozone can be effectively delivered to a caries lesion to kill S. mutans.

5. To determine if S. mutans can be killed by ozone where the concentrations are

validated.

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3. Materials and methods

3.1 Bacterial species and culture

The bacterial species used in this study was Streptococcus mutans strain ATCC 10449

(serotype c).

A stock culture of the bacteria was transferred to Wilkins-Chalgren broth media (WC,

Difco Laboratories. Sparks) and then incubated at 37°C in aerobic (ambient) atmosphere.

After 24 hours, an aliquot of this culture was inoculated to a fresh W-C tube, to an

approximate concentration of 108 colony forming units (CFU) /ml using McFarland standards

(equivalent to ~108 CFU/ml).

3.2 Ozone generators

The ozone generator used for this study was the Curozone X4® (Kavo, Biberach/Riß,

Germany) generator (Fig. 1). The device is designed to generate ozone either using

atmospheric oxygen or with supplemental oxygen delivered with an attached oxygen tank to

yield higher ozone concentration. According to the manufacturer the generated ozone

concentration is 2100 ppm, ±10%, delivering 65µmol ozone/min. The exposure time can be

set from 5 to 60 sec. The ozone is delivered through a hand-piece fitted with disposable

silicone tips of variable sizes as appropriate to the treatment surface, designed to seal the

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surface, otherwise the device stops working. After the exposure time there is a two sec

vacuum/ scrubbing cycle to eliminate any remaining ozone in the system thus preventing

escape of ozone to the ambient atmosphere (Fig. 2).

The ozone generator OL80A (Ozone Services, Yanco Industries, Burton, BC). (Fig.

3), was used to expose the bacteria in the Mouse Exposure Chamber (ESE Design Center,

Department of Environmental Sciences and Engineering, Gillings School of Global Public

Health. University of North Carolina at Chapel Hill) (Fig. 4) to atmospheres of either 2 or 20

ppm of ozone. The OL80W (Fig. 5, located at the Environmental Sciences and Engineering

Laboratory located at Gillings School of Global Public Health at the University of North

Carolina at Chapel Hill) was used to expose the bacteria in the Modular Incubator Chamber

(Billups-Rothenberg, Inc. Del Mar, CA) (Fig. 6) to atmospheres of either 200 or 2000 ppm of

ozone.

3.3 Effect of ozone on human dental specimens.

Thirty-six extracted human third molars were used as dental specimens. The teeth

were debrided and examined to ensure that they were free of defects (caries, fractures,

cracks). The crowns were separated from the roots using an Isomet diamond saw (Buehler

Ltd., Lake Bluff, IL) under running water to obtain 6 x 4 mm slabs. The specimens were

ground mechanically (Ecomet 3, Buehler Ltd.) under running water using a 600-grit silicon

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carbide (SiC) paper to obtain flat dentin surface (Fig. 7). Single preparations were made on

the specimens with a #2 bur at high speed with water refrigeration to create a cavity of 1 mm2

that would harbor bacteria; they were then sterilized with steam autoclave. The specimens

were then inoculated with serial ten-fold dilutions of the bacteria culture 108 to 106 CFU/ml

and incubated at 37°C for 24 hours. The specimens were exposed to ozone generated by the

Curozone X4® which produces concentrations up to 2100 ppm, according to the manufacture.

This was accomplished using the Curozone handpiece fitted with 3 mm silicone tips. This

completely surrounded the “inoculated lesion”, creating a sealed ozone atmosphere at the

treatment site. Fresh tips were used with each treatment to prevent cross-contamination. The

treatment timing was begun automatically by the instrument once the desired ozone

atmosphere was achieved.

The specimens were randomly divided in three groups: (n=12)

1. Low ozone group: Exposed to ozone generated using ambient air for 60 seconds.

2. High ozone group: Exposed to ozone generated using pure oxygen for 60 seconds.

3. Control group: Exposed to ambient air for 60 seconds.

After the exposure to air or ozone, dentin shavings were collected from the

circumferential cavity walls to a 1.4 mm depth into 1 ml sterile bacterial transport media

containing glass beads to facilitate dispersion of the bacteria clumps with vortexing, by using

sterilized #4 carbide burs at low speed (NSK Ultimate 450K, Nakanishi Inc. Japan). A new

sterile bur was used for each specimen to prevent over heating of dentinal walls and cross-

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contamination.

Serial ten-fold dilutions of the vortexed transport media were used to determine the

bacteria counts from the dentin shavings obtained. The diluted solutions were spiral plated

(Spiral Systems Inc., Cincinnati, OH) on Trypticase™ Soy Agar with 5% Sheep Blood.

(Becton, Dickinson and Company. Sparks, MD). After 24 hours of incubation at 37°C, the

resulting colonies were counted using a ProtoCol automatic plate counter (Spiral Systems

Inc., Cincinnati, OH) and Protocol software. Data were calculated as CFU/ml of the original

transport medium. To normalize the CFU/ml, descriptive statistics (means and standard

deviations) were performed on log transformed data.

3.4 Effect of ozone on bovine dental specimens

Thirty six extracted bovine teeth were used. The teeth were debrided and examined to

ensure that they were free of defects (caries, fractures, cracks). The crowns were separated

from the roots using an Isomet diamond saw (Buehler Ltd., Lake Bluff, IL) under running

water to obtain 10 x 6 mm slabs. The specimens were ground mechanically (Ecomet 3,

Buehler Ltd.) under running water using a 600-grit SiC paper to obtain a flat dentin surface,

(Fig. 8). Single preparations of 1.4 mm2 were made on the specimens with a #4 bur at high

speed with water refrigeration to create a cavity that would harbor bacteria. They were then

sterilized with steam autoclave. The specimens were then inoculated with ten fold dilutions of

the bacteria culture 108 to 106CFU/ml and incubated at 37°C for 24 hours.

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The results of Experiment 1 show that the recovered log10 CFU obtained from low

ozone, high ozone and control groups did not differ significantly among the three groups

results. It was therefore decided to compare the results of the two test groups: high ozone and

low ozone delivered using handpiece of the Curozone X4® fitted with 3 mm silicon tips as

described above for the human teeth.

The specimens were randomly divided in two groups: (n=18)

1. Low ozone control group: Exposed to ozone generated using ambient air for 60

seconds.

2. High ozone: Exposed to ozone generated using pure oxygen for 60 seconds.

After the exposure to ozone generated from ambient air or using pure oxygen, dentin

shavings were collected from the circumferential cavity walls of diameter to 1.8 mm depth

into 1 ml sterile bacterial transport media containing glass beads to facilitate dispersion of the

bacteria clumps with vortexing, sterilized #6 carbide burs at low speed. A new sterile bur was

used to prevent over-heating of dentinal walls and cross-contamination.

Serial ten-fold dilutions of the vortexed transport media were used to determine the

bacteria counts from the dentin shavings obtained. The solution was spiral plated on

Trypticase™ Soy Agar with 5% Sheep Blood (WC, Difco Laboratories. Sparks). After 24

hours of incubation at 37°C, the resulting colonies were counted using a ProtoCol automatic

plate counter and Protocol software as described above.

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3.5 Bactericidal activity as loss of recoverable CFU after exposure of S. mutans inoculated agar surfaces to defined atmospheres of ozone

After showing the inability of the Curozone X4 to effectively inhibit S. mutans on

human and bovine roots at purported levels of ozone of 2100 ppm for 1 min, it was decided to

determine if the target strain of bacterium (S. mutans ATCC 10449) is, in fact, susceptible to

ozone. To optimize delivery to the target, the bacteria were spread on a Trypticase™ Soy

Agar with 5% Sheep Blood surface to allow uniform delivery of ozone in a contained

atmosphere. These inoculated plate surfaces were treated by directly exposing the uncover

plates to specific ozone atmospheres. Determination of the effective killing dose of ozone was

accomplished by varying the exposure atmosphere to include 2, 20, 200 and 2000 ppm of

ozone for 4 min under laboratory conditions using a different ozone generator. In addition, the

2 and 20 ppm groups were tested with the Petri dish covers in place to determine if the cover

would be a physical barrier to ozone access to the inoculated surface.

The experiments were performed under defined conditions at the Environmental

Sciences and Engineering Laboratory located in the Gillings School of Global Public Health

at the University of North Carolina at Chapel Hill.

To determine the bactericidal activity of ozone, an overnight culture (early

exponential) of S. mutans was adjusted to a suspension of 106 CFU/ml and 49.2 l was spiral

plated to replicates of seven SBA plates. Six plates were treated by varied exposures to

atmospheres of ozone and one was left untreated as control. The 2000 and 200 ppm ozone

atmosphere groups were exposed using the OL80A ozone generator into the Modular

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Incubator Chamber. The 2 and 20 ppm ozone atmosphere groups were exposed either with or

without the Petri dish cover in place using the OL80W ozone generator into the Mouse

Exposure Chamber. The larger size of this chamber permitted the simultaneous treatment of

multiple plates in a single plane.

The distribution of the groups was as follows:

1. Uncovered replicate plate exposed to 2000 ppm of ozone for 4 min

4. Replicate plate exposed to 20 ppm of ozone for 4 min with the Petri dish cover in

place to determine if the cover would be a physical barrier to ozone

6. Replicate plate exposed to 2 ppm of ozone for 4 min with the Petri dish cover in

place to determine if the cover would be a physical barrier to ozone

7. Untreated replicate plate as a control for determining recoverable CFU.

Following treatments the replicate plates were incubated in an anaerobic chamber

(10% H2 – 5% CO2 – 85% N2) at 37°C for 18 hours to permit development of the CFU. After

this period the resulting CFU were counted using a ProtoCol automatic plate counter. The log

transformed data were treated as described above. The experiments exposing uncovered plates

to the various atmospheres were repeated on three independent occasions. Treatment of

covered plates with 2 and 20 ppm ozone atmosphere was only done once.

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3.6 Influence of ozone exposure of S. mutans suspended in liquid in microtiter plate wells on bacterial viability.

Our previous experiments with S. mutans in the artificial cavity preparations in either

human or bovine dentin slabs determined that exposure to ozone delivered directly with the

hand piece did not result in demonstrable reduction in viability of the bacteria target even at

the highest concentration (2,100 ppm) for up to 60 sec. Longer treatments were technically

challenging with the small dentin slabs and there relatively irregular surfaces. It was also

possible that the failure to kill was due to the competitive inactivation of the ozone by the

dentin. The aqueous environment of the suspended bacteria in the “cavity” could also have

limited accessibility of the ozone to the target bacteria. In the next set of experiments, a more

uniform approach for testing the effects of ozone atmospheres on the viability of S. mutans in

an aqueous environment was sought. It was determined that the 3 mm silicone tip fit formed a

tight seal with the well of a 96-well microtiter plate. This permitted the creation of individual

ozone atmospheres in the micro-format of each well. A liquid volume containing the target

bacteria could be uniformly delivered to the bottom of each well and exposed to a define

ozone atmosphere. After exposure the treated bacteria could be resuspended in culture

medium and incubated to permit growth. Total killing of the bacteria would result in a failure

to grow (no change in optical density). Logarithmic killing of the bacteria would result in

delays in attaining discernible growth and consequent reductions in attained optical densities

when compared to untreated controls. The effectiveness of Curozone X4® on bacteria

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suspended in liquid was therefore tested by comparing the post-treatment growth as optical

density (mO.D.at λ610 nm) 18 to 24h after exposure to 15, 30, 45 and 60 seconds of ozone

with that of untreated controls.

A 96-well microtiter plate (Fig.9) was used to receive ten microliter (l) of 106

CFU/ml of bacteria to be treated with ozone through the Curozone X4®unit using a 3 mm

silicone tip in high and low ozone modes. The controls consisted of two groups of 200l of

media without bacteria, one group was treated for 1 min and the other was left untreated. The

positive control was 10l of the bacteria culture that was not treated.

Each bacteria and control group, with the exception of the untreated media group, was

divided in 2 groups of 4 wells each. One received the low ozone treatment and the other high

ozone treatment.

The group distribution was as follows:

1. Media untreated. 200l.

2. Media treated for 1 min. 200l.

3. Bacteria untreated. 10l of 106 CFU/ml.

4. Bacteria treated for 15 sec. 10l of 106 CFU/ml..

5. Bacteria treated for 30 sec. 10l of 106 CFU/ml.

After treatment, the 10l aliquots of bacteria were resuspended in 200l of fresh W-C

(28)

medium broth and incubated in an anaerobic chamber at 37oC. The optical densities (λ610

nm) of the quadruplicate samples were determined after 18 hours by using a Vmax kinetic

microplate reader and companion SoftMax Pro 3.1 software (Molecular Devices, Sunnyvale

CA). Data were expressed as mOD and means and standard deviations determined on

replicate wells. Each experiment was independently repeated at least three times.

3.7 Effect of ozone in S. mutans suspended in media in exposures times of 1, 2, 3 and 4 min

This experiment was designed to determine ozone effectiveness on bacteria in media

after different exposures times ranging from 1 to 4 min.

A 96 well microtiter plate was used to receive ten microliter (l) of 106 CFU/ml of

bacteria to be treated with ozone through the Curozone X4® unit using a 3 mm silicone tip in

high and low ozone modes in exposures time of 1, 2, 3 and 4 min. The controls consisted of

two groups of 200 l of medium without bacteria, one group was treated for 1 min and the

other was left untreated. The positive control was 10l of the bacteria culture that was not

treated.

divided in 2 groups of 4 wells. One received the low ozone treatment and the other high ozone

treatment.

(29)

1. Media untreated. 200l

2. Media treated for 1 min. 200l

3. Bacteria untreated 10l of 106 CFU/ml.

4. Bacteria treated for 1 min. 10l of 106 CFU/ml.

medium broth and incubated in an anaerobic chamber at 37oC. The optical densities (λ610

nm) of the quadruplicate samples were determined after 18 hours by using a Vmax kinetic

microplate reader and companion SoftMax Pro 3.1 software (Molecular Devices, Sunnyvale

CA).

3.8 Effect of ozone in S. mutans suspended in media in concentrations of 106, 105, 104 and 103 for 1 min

This experiment was designed to determine ozone effectiveness on titrated bacteria in

concentration of 106,105,104 and 103 CFU/ml in media for 1 min.

This experiment was designed to determine ozone effectiveness on titrated bacteria in

concentration of 106,105,104 and 103 CFU/ml in media for 1 min. A 96 well microtiter plate

(30)

was used to receive ten microliter (l) of 106 CFU/ml of bacteria to be treated with ozone

through the Curozone X4®unit using a 3 mm silicone tip in high and low ozone modes. The

controls consisted in two groups of 200 l of media without bacteria, one group was treated

for 1 min and the other was left untreated. The positive control was 10l of the bacteria

culture that was not treated.

divided in two groups of four wells. One received the low ozone treatment and the other high

ozone treatment.

1. Media untreated. 200l

2. Media treated. 200l

3. Bacteria untreated 10l of 106 CFU/ml.

medium and incubated in an anaerobic chamber at 37oC. The optical densities (λ610 nm) of

the quadruplicate samples were determined after 18 hours by using a Vmax kinetic microplate

reader and companion SoftMax Pro 3.1 software (Molecular Devices, Sunnyvale CA).

(31)

3.9 Effect of ozone in S. mutans suspended in media and on agar under controlled conditions

The ozone generator OL80W was used to expose the bacteria in the Modular

Incubator Chamber at Environmental Sciences and Engineering Laboratory located at Gillings

School of Global Public Health at the University of North Carolina at Chapel Hill.

This experiment was designed to test the effectiveness of exposing S. mutans in media

and on a surface to ozone gas, at the same time for four min in an atmosphere of 2000 ppm

ozone. To accomplish this objective two groups were designed:

Media Group: A total of 15 wells of a 96 well microtiter plate were inoculated with

10l of 103 CFU/ml. Five wells were inoculated with 200l of media serving as controls.

(Fig.10). Two sets were prepared one was treated and the other left untreated as control.

Agar Group: For this group two agar plates with 49.2l from a culture 106 CFU were

spiral plated on Trypticase™ Soy Agar with 5% Sheep Blood. One was treated with ozone

and the other left untreated as control.

After being exposed to ozone at 2000 ppm for four min, the 10l suspension of the

treated bacteria in the media group was brought to a volume of 210l with fresh W-C medium

and incubated in an anaerobic chamber at 37oC. The optical densities (λ610 nm) of the

quadruplicate samples were determined after 18 hours by using a Vmax kinetic microplate

reader and companion SoftMax Pro 3.1 software (Molecular Devices, Sunnyvale CA).

(32)

4. Results

4.1 Effect ozone in S. mutans on human dental specimens

Results are presented in Table 2. The The mean recoverable CFU/ml obtained from

low ozone (5.25 ± 0.87), high ozone (5.47 ± 0.92) and control (5.58 ± 0.67) groups did not

differ significantly among the three groups. (Kruskal-Wallis test, p=0.58).

During the experiment it was noticed the handling difficulties of the Curozone X4®,

keeping the seal of the silicone tip turn to be problematic, changings in pressure and holding

angle of the hand-piece lead to seal loss, also the irregularities of the tooth surface made the

dentin shaving proccess challenging. Due to this inconvenient it was decided to utilize bovine

teeth to minimize the mechanical problems found with human teeth.

4.2 Effect ozone in S. mutans on bovine dental specimens

Results are reported in Table 3. There was no statistically significant difference

between the mean of recovered log10 CFU obtained from low ozone control (5.37 ± 0.71) and

high ozone (5.32 ± 0.82) (Wilcoxon test, p=0.53).

Having a larger tooth surface did not prevent the mechanical problems explained in

(33)

Curozone X4® is not delivering ozone to the lesion site in a concentration that is sufficient to

kill or that the test bacterial strain is inherently resistant to ozone. A different approach is

proposed, to test the antibacterial effect of ozone under control conditions at the

Environmental Sciences and Engineering Laboratory located at Gillings School of Global

Public Health at the University of North Carolina at Chapel Hill.

4.3 Effect ozone in S. mutans suspended in media under controlled conditions

Results are reported in Table 4.The plates exposed to 2000 and 200 ppm had no

recoverable colony forming units (total inhibition). The 20 ppm group that was left uncovered

showed 99.99% bacteria inhibition (1.59 log10 CFU/ml recovered), while the covered group

showed 99% bacteria inhibition (3.55log10 CFU/ml recovered). The 2 ppm group that was left

uncovered showed 99.99% bacteria inhibition (2.81 log10 CFU/ml recovered), while the

covered groups showed 91.41% bacteria inhibition (4.58 log10 CFU/ml recovered). The

untreated control group showed a bacteria value of 5.64 log10 CFU/ml. Logarithmic

reduction in recoverable CFU greater than 1 compared to control was considered significant.

These results show the antibacterial effect of ozone on S. mutans while spread on a

surface, during the test another agar plate was spot assay with 10l of a titration of S. mutans,

L. acidophilus and E. faecalis from 109 to 103 and exposed to ozone at 2000 ppm for 4 min, resulting in complete killing of the bacteria. This experiment was made in collaboration with

Dr. Marcos Ximenes.

(34)

A new experiment for the Curozone X4® is designed. The tooth model could bring

limitations in the recovery of the treated bacteria which can diffuse through the dentin tubules

and become unreachable to ozone; in this approach a 96 microtiter well was used to avoid the

bacteria recover process with the tooth model.

4.4 Effect ozone in S. mutans suspended in media for 15, 30, 45 and 60 s

Results are reported on Table 5. No killing was observed. Bacteria counts are not

different among the high ozone and low ozone groups (Wilcoxon rank sum test, p=0.42).

Being unable to reduce the bacteria recover in different exposure times a new

experiment was run increasing the time for 1, 2, 3 and 4 min.

4.5 Effect ozone in S. mutans suspended in media for 1, 2, 3 and 4 min

different among the high ozone and low ozone groups but they are significantly different

compared to media control groups (Wilcoxon rank sum test, p=0.53).

Being unable to reduce the bacteria recover in exposures up to 4 min, a new

experiment was designed titrating the bacteria concentration one log from 106 to 103.

4.6 Effect ozone in S. mutans suspended in media in concentrations of 106, 105, 104 and 103

(35)

different among the high ozone and low ozone groups but they are significantly different

compared to media control groups (Wilcoxon rank sum test, p=0.56).

These results demonstrate the inability of the Curozone X4® to effectively reduce the

CFU/ml of bacteria on suspension. A new experiment testing the ozone effect under control

conditions was run exposing bacteria in a microtiter plate and a spiral plate and the same time

to 2000 ppm for 4 min.

4.7 Effect ozone in S. mutans in media and on agar

Results are shown on Table 8. The bacteria density of the treated (424 mO.D. λ610

nm) and untreated group (432 mO.D. λ610 nm) showed no bacteria killing.

For the agar group, the plate that was treated with ozone gas showed 100% bacteria

inhibition, the untreated control group showed 5.63 log10 CFU/ml. Bacteria counts are

different among the 3 mutually exclusive groups of non-treated, ozone treated, treated media.

(Wilcoxon. p<.0001).

(36)

5. Discussion

The association between plaque levels of S. mutans and caries is well described35-38 .

Bowden et al (1990) found the strongest association when plaque was removed from single

occlusal fissures. Seventy-one percent of the carious fissures had S. mutans accounting for

more than 10% of the viable flora, whereas 70% of the fissures that were caries free had no

detectable S. mutans. Sixty-five percent of the pooled plaque samples from the children with

rampant caries had S. mutans accounting for more than 10% of the viable flora, whereas 40%

of the pooled samples from children that were caries free had no detectable S. mutans35.

Therefore methods to eliminate or inhibit S. mutans in the oral cavity must be the main

objective in caries prevention. Ozone is considered a strong oxidizer of the cell walls and

cytoplasmatic membranes of bacteria and is considered a potent bactericidal, antiviral, and

antifungal agent39. It is important to point out that this statement is based on the use of ozone

in blood not gassing ozone to a microfilm.39, 40 This is a common finding in the literature,

extrapolating the use and effect of systemic ozone dissolving it in the bloodstream, to the

dental field.18, 39 These articles explain the indications and mechanism of action of ozone

dissolved in blood but there is no reference to the effect in the oral cavity. Therefore the real

ability of ozone to kill cariogenic bacteria is yet to be determined.

(37)

grow to form biofilms. Biofilms have been defined as matrix embedded microbial

populations, adherent to each other and/or to surfaces or interfaces.2, 41, 42 This is especially

true in the oral cavity which is permanently in wet conditions. One property of biofilms is the

protection from host defenses, desiccation etc. by production of extracellular polymers to

form a functional matrix2 this is a key factor understanding the effect of antimicrobial agents,

they have to be able to reach the bacteria through the biofilm. There is evidence that bacteria

living in biofilms as dental plaque on tooth surfaces are generally more resistant to

antimicrobial agents than bacteria in batch culture normally used for “in vitro” susceptibility

testing43. The bacteria culture used in this study was grown in a sucrose free media; therefore

the bacteria could not secrete a glucan layer that would allow creating a protective biosphere.

The antibacterial effect of ozonated water and ozone bubbled in liquid has been

extensively published. 20, 23, 39, 40, 44 There are numerous publications that claim an antibacterial

effect ozone in vivo7, 12 and in vitro8, 9, 11, 13, 14, 16, 17, 20 but several authors have reported the inability of gas ozone to reduce the number of bacteria as a result of treatment 4, 22, 29, 30, 32, 33

which are in accordance with the results obtained in this investigation when using the

Curozone X4®on both root surfaces and microtiter plates and the ozone generator OL80A on

the microtiter plate. Reviewing the literature that supports the use of ozone in dentistry it was

found that not all the cited “in vitro” studies tested ozone in the same conditions.11, 13, 14, 20

Baysan et al. (2000) tested the effect of ozone gas and ozonated water on S. mutans and S.

sobrinus, the bacteria was collected from primary carious lesions on freshly extracted teeth;

(38)

they concluded that ozone gas application for a period of 10 s was capable of reducing the

numbers of S. mutans and S. sobrinus on saliva-coated glass beads “in vitro”. The article by

Nagayoshi et al. (2005) on S. mutans tested the effect of ozone on dental plaque samples from

human subjects showing that S. mutans was killed immediately when exposed to ozone water;

however there was no exposure to ozone gas in this study. Johansson et al. (2009) showed

99.9% killing of S. mutans in saliva after being exposed to ozone gas for 60 s. It is important

to point out that the ozone generator used in this investigation KaVo HealozoneTM 2130C

(Curozone; KaVo Biberach/Riss, Germany) was modified to bubble the bacteria culture. The

device was not used as proposed by the clinical protocol in the oral cavity.

In contrast, there are other “in vitro” studies showing the inability of ozone gas to

effectively reduce the bacteria number 22, 29, 30, 32, 45 Hems et al. (2005) compared the effect of

sodium hypochlorite (NaOCl ) and ozone gas generated by Purezone (Ipswich, UK). They

concluded that “Ozone had an antibacterial effect on planktonic E. faecalis cells and those

suspended in fluid, but little effect when embedded in biofilms. The antibacterial efficacy of

ozone was not comparable with that of NaOCl under the test conditions used”. In 2007 Muller

et al. published an article where ozone gas, photodynamic therapy, chlorhexidine in 0.2 and

2% and NaOCl in 0.5 and 5% were applied to A. naeslundii, V. dispar, F. nucleatum, S.

sobrinus, S. oralis and C. albicans biofilms on bovine teeth. The HealOzone 2130C device generator was used. They found that “only the 5% sodium hypochlorite solution was able to

totally eliminate the microorganisms in the biofilm. The observed reduction of viable counts

(39)

by vacuum-ozone application and PDT was less than one log10 step. Under the conditions of

the study, gasiform ozone and PDT had a minimal effect on the viability of microorganisms

organized in a cariogenic biofilm”. Polydorou et al. (2012) tested the HealOzone 2130C

device by inoculating S. mutans and L. cassei in artificially made cavities in caries-free third

molars and then treat them for 60 s with ozone. The cavities were filled with composite. After

4 and 8 weeks, the bacteria number was determined. The conclusion brought is the exposure

of ozone through the “HealOzone 2130C device for 60 s can provide some antibacterial

treatment against S. mutans even after 8 weeks. However, an elimination of the

microorganisms seems not to be possible. L. cassei was more resistant to ozone. Although

ozone exerts a significant antibacterial effect against S. mutans, it is probably not enough as

the only antibacterial method, during the fillings therapy”.30 The article published by Estrela

et al (2007) tested the effect of ozonated water, gaseous ozone (PXZ3507; Eaglesat

Tecnologia em Sistemas Ltda., Sao Jose dos Campos, SP, Brazil), sodium hypochlorite

(2.5%) and chlorhexidine (2%) on E. faecalis inoculated in human roots. They concluded that

“The irrigation of infected human root canals with ozonated water, NaOCl, chlorhexidine and

the application of gaseous ozone for 20 min was not sufficient to inactivate E. faecalis”. Iman

et al. (2008) tested the effect of 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine

(CHX) and ozonated water on E. faecalis and C. albicans biofilms. They found that NaOCl

succeeded in complete degradation of the biofilm. This effect was less with CHX and even

less with ozonated water. Their conclusion is that the antimicrobial effectiveness of 5.25%

(40)

NaOCl was reinforced, whereas that of ozonated water was questionable.

Previous studies with positive results have been presented in Europe but there are

several studies that question the effectiveness of ozone as an anticariogenic agent. A

Cochrane Database of Systematic Reviews 34 on three clinical studies reported that the

analyses of all the studies were conducted at the level of the lesion, which is not independent

of the person, for this reason pooling of data was not appropriate or attempted. Few secondary

outcomes were reported, but one trial reported an absence of adverse events. Also, masking of

participants was absent (as no placebo was provided), and there are doubts about the

randomization. Therefore, the Cochrane study concluded that given the high risk of bias in the

available studies and lack of consistency between different outcome measures, there is no

reliable evidence that application of ozone gas to the carious lesion stops or reverses the

process30,46.

Several investigations show that different killing methods have demonstrated dose

effectiveness.29, 47 Gomes et al. (2001) compared sodium hypochlorite (NaOCl) (0.5%, 1%,

2.5%, 4% and 5.25%) and chlorhexidine (CHX) liquid and gel at (0.2%, 1% and 2%). it was

concluded that all irrigants were effective in killing E. faecalis, but at different times. CHX in

the liquid form at all concentrations tested (0.2%, 1% and 2%) and NaOCl (5.25%) were the

most effective irrigants. However, the time required by 0.2% CHX liquid and 2% CHX gel to

promote negative cultures was only 30 s and 1 min, respectively in contrast NaOCL 5.25%

required less than 30 s to produce 100% inhibition growth. 47Their conclusion was that all

irrigating solutions had antibacterial activity but it effectiveness was depended on its

(41)

concentration. The results in the present study show that ozone is not dose dependent, there

was no effective bacteria killing either after an exposure of 15 seconds or 4 minutes in the two

different experimental designs. In the root experiments, the results were similar among the

low and high ozone modes compared to the control while exposing 7l of 106 CFU/ml for up

to 60 sec; the microtiter experiment show similar result where effective killing could not be

achieved in exposition that were form 15 to 4 min and the results were not different from the

control.

The present in vitro study could not demonstrate any reduction in the number of S.

mutans after being treated with gasiforme ozone using the Kavo Curozone X4® generator in exposures that went from 15 sec to 4 min; the results were similar on roots as well as in

microtiter plates. It is interesting to mention that while using the Curozone X4® it was found

that the pressure required to keep the seal of the silicone tip to the surface being treated was

minimal, when increasing the pressure, the device stopped working making this procedure

very sensitive and not easy to perform even on the bench in the laboratory, so we presume

that clinically would be even more difficult to obtain good sealing and consequently easy

application. Even though some publications suggest that “It is recommended to deliver ozone

be under pressure into a lesion by pressing the delivery tube onto the carious surface so that it

can penetrate the lesion”.15, 17

It was also noticed that after the ozone exposures some biofilms

were desiccated and some other were wet; but the bacteria recovery did not show any

difference compared to the control, this finding is in accordance with the study published by

(42)

Hems et al. (2005). It has been published that “ozone will react immediately with the

reductants in culture media, and the authors did not bubble the ozone into the biofilm”.15, 16

During the present investigation the bubbling effect was not seen when the ozone was applied

to bacteria in media. Actually while using the Curozone X4®when pressure was applied the

device immediately stopped the ozone delivery.

The ozone concentrations tested were 2 and 20 using the ozone generator OL80A in

the Mouse Exposure Chamber; for the 200 and 2000 ppm the ozone generator OL80W in the

Modular Incubator Chamber for exposure time of 4 min. It is important to mention that the

samples were exposed longer that the mentioned times, the chamber took around 20 min to

reach the concentration levels and then the 4 min were counted, therefore the exposure time to

ozone was longer but it is important to emphasize that the effect on bacteria in the microtiter

plate was still negative. The inability of ozone gas to diffuse in liquid has been proposed by

different authors.22, 48 In 1976 Hoigné and Bader stated that “Ozone is a selective oxidant and

affects only certain compounds but when it dissolves in water, it becomes highly unstable and

rapidly decomposes through a complex series of chain reactions”. It has also been stated that

the stability of ozone in water is low and the ozone dissipates very quickly at room

temperature.49

Separate experiments in collaboration with Dr. Marcos Ximenes were conducted at the

same time as the spiral plated assays described above. Instead of spiral plating 49.2ml

volumes, 10l volumes of S. mutans, Lactobacillus acidophilus and Enterococcus faecalis in

concentration ranging from 108 to 103 CFU/ml were delivered as drops on the agar surface

(43)

prior to exposure to atmospheres of 2, 20, 200 and 2000 ppm of ozone for 4 min. This

simulated the volumes in the microtiter plate wells. As with the spiral plate studies with S.

mutans, the results obtained showed 100% killing at 2000 and 200 ppm and logarithmic reductions in 20 and 2 ppm atmospheres with all three test species. In addition an experiment

exposing 10l of 106 CFU/ml of S. mutans on the bottom of an agar free Petri dish to 2000

ppm of ozone for 4 min, showed 100% killing (Unpublished data). This may suggest that

ozone gas does not equilibrate with the atmospheric air into a small space such as a well in a

microtiter plate as a result it is no capable to have an antimicrobial effect. The results obtained

in the experiment conducted by Dr. Marcos Ximenes show that ozone can effectively kill

bacteria when it has access to the microorganism and while being on a surface in low volume

of liquid (unpublished data) and probably ozone is ineffective in larger liquid amounts.22, 48

These results may explain why the Curozone X4 could not kill bacteria, the ozone generated

by the instrument was not able to adequately displace the air either in the artificial lesion

preparation in the dentin slab or in the microtiter plate to achieve bactericidal concentrations

of ozone in the target bacterial suspension.

The negative results of this and other in vitro investigations along with the fact that

bacteria living in biofilms as dental plaque on tooth surfaces are generally more resistant to

antimicrobial agents than bacteria in batch culture normally used for in vitro susceptibility

testing43; raise questions about the antimicrobial capacity of the Curozone X4 for clinical use.

In a communication received by the manufacture it was assured that the device used for this

(44)

investigation had been tested by the quality control department of the company and was

properly working before being sent to us. (See appendix A).

The results of the present study suggest that ozone in controlled conditions is not

capable to effectively reduce the bacteria number inoculated in media in long exposures when

placed in a microtiter plate. In contrast ozone was able to reduce the bacteria number when it

had adequate access to the bacteria (Petri dish (100 mm diameter) vs. microtiter well (6 mm

diameter) vs. the ~ 1 mm diameter simulated lesion in the dentin slab. The latter result

confirms the finding of previous studies that ozone gas is effective in killing S. mutans.25, 27

(45)

6. Conclusions

Without the obvious limitations that exist in a clinical environment, it can be

concluded that the Curozone X4® generator is not capable to effectively reduce the number of

S. mutans in exposure times up to 4 min.

When the bacteria exposure is in an open surface, ozone at 2000 ppm for 4 min can

completely kill S. mutans. Concentrations of ozone as low as 2 ppm result in biologically

significant (exponential) killing of S. mutans.

This study suggests that Curozone X4® generator is not capable of delivering ozone in

(46)

7. Tables

Table 1. Indication of the ozone therapy in medicine.19

Indications Mechanisms

External ulcers and skin lesions Disinfection, wound cleansing and improved

wound healing

Arterial circulatory disorders Activation of red blood cell metabolism

with an improvement of oxygen release Activation of reactive oxygen species and radical scavengers

Immunodeficiency and

immunodysbalance: Activation of immunocompetent cells with

Chronic forms of release of cytokins such as interferons and

hepatitis B and C interleukins.

Supportive therapy in cancer patients

Supportive therapy in Modulation of the immune system

rheumatoid arthritis Increase of antioxydative capacity

by activation of biological antioxidants

Inflammatory condition such as: Antiinflammatory effect

Knee arthrosis Activation of antioxidative enzymes as radical

Gonarthrosis scavengers

Traumatic knee disorders Activation of immunocompetent

(47)

Table 2. Number of bacteria recovered after the different treatment protocols applied with the Kavo Curozone X4® ozone generator to human dentin specimens for 60 s (log10 CFU)

Treatment protocol (n=36) log10 CFU (Mean ± SD)

Low ozone1 5.25 ± 0.87

High ozone2 5.47 ± 0.92

Control 3 5.58 ± 0.67

(Kruskal-Wallis test, p=0.58)

1. Exposed to ozone in ambient air for 60 seconds. n=12

2. Exposed to ozone in oxygen using the attachedtank for 60 seconds. n=12 3. Exposed to ambient air for 60 seconds. n=12

(48)

Table 3. Number of bacteria recovered after the different treatment protocols applied with the Kavo Curozone X4® ozone generator to bovine dentin specimens for 60 s (log10 CFU)

Treatment protocol (n=36) log10 CFU

Low ozone control1 5.37 ± 0.71

High ozone2 5.32 ± 0.82

(Wilcoxon rank sum test, p=0.53)

1. Exposed to ozone in ambient air for 60 seconds. n=18

2. Exposed to ozone in oxygen using the attachedtank for 60 seconds. n=18

(49)

Table 4. Number of bacteria recovered and killing rates after exposing S. mutans on agar (log10 CFU/ml) to ozone in the Modular Incubator Chamber at different concentrations for 4 min in covered and uncovered Petri dishes.

After 24

hours Control 2 ppm

2 ppm

cover on 20 ppm

20 ppm

cover on 200 ppm 2000 ppm mean 5.64 2.81 4.58 1.59 3.55 0 0

log10

reduction 2.83 1.06 4.05 2.09 5.64 5.64

logarithmic reduction in recoverable CFU greater than 1 compared to control was considered significant.

(50)

Table 5. Bacterial growth as optical density (mO.D. at λ610 nm) attained following incubation at 37oC for 18 hr after the different treatment protocols applied with the Kavo Curozone X4® ozone generator on 10l of 106 CFU/ml of S. mutans

106 CFU 106 CFU 106 CFU 106 CFU 106 CFU untreated 15 sec 30 sec 45 sec 1 min

Low mean 281 278 289 282 299

Ozone sd 38 45 9 7 13

High mean 312 308 297 302 306

Ozone sd 14 2 7 15 4 (Wilcoxon rank sum test, p=0.42)

(51)

Table 6. Bacterial growth as optical density (mO.D. λ610 nm) attained following incubation at 37oC for 18 hr after the different treatment protocols applied with the Kavo Curozone X4® ozone generator on 10l of 106 CFU/ml of S. mutans

106_{CFU 10}6_{CFU 10}6_{CFU 10}6_{CF 10}6_CFU untreated 1 min 2 min 3 min 4 min

Low mean 229 257 251 244 245

Ozone sd 13 8 15 18 20

High mean 268 256 233 257 236

(52)

Table 7. Bacterial growth as optical density (mO.D. λ610 nm) attained following incubation at 37oC for 18 hr after the different treatment protocols applied with the Kavo Curozone X4® ozone generator on 10l of a bacteria titration from 106 to 103 CFU/ml of S. mutans

106_{CFU 10}6_{CFU 10}6_{CFU 10}6_{CFU 10}6_CFU untreated 1 min 2 min 3 min 4 min

Low mean 267 263 251 244 200

Ozone sd 10 9 15 18 19

High mean 265 266 233 257 168

(53)

Table 8. Bacterial growth as optical density (mO.D. λ610 nm) recovery in media and recovery (log10 CFU/ml) on agar attained following incubation at 37oC for 18 hr after the different treatment protocols applied with the ozone generator

OL80W in the Modular Incubator Chamber at 2000 ppm for 4 min

At 24 hours

Non treated bacteria in

media1

Ozone treated bacteria in

media1

Non treated bacteria in

agar2

Ozone treated bacteria in

agar2

mean 432 424 5.63 0

SD 108 158